Recent vaccination strategies make use of viruses or virus-like-particles (VLPs) to enhance the immune response towards antigens. For example, WO02/056905 demonstrates the utility of VLPs as a carrier to present antigens linked thereto in a highly ordered repetitive array. Such antigen arrays can cause a strong immune response, in particular antibody responses, against the linked antigen and are even capable of breaking the immune system's inherent tolerance towards self antigens. Such antigen arrays are therefore useful in the production of vaccines for the treatment of infectious diseases and allergies as well as for the efficient induction of self-specific immune responses, e.g. for the treatment of cancer, rheumatoid arthritis and various other diseases.
As indicated in WO02/056905 capsid proteins of bacteriophages are particularly suited as antigen carrier. They have been shown to efficiently self-assemble into VLPs upon expression in a bacterial host (Kastelein et al. 1983, Gene 23:245-254; Kozlovskaya et al. 1986, Dokl. Akad. Nasik SSSR 287:452-455). Moreover, capsid proteins of bacteriophages such as derived from fr (Pushko et al. 1993, Protein Engineering 6(8)883-891), Qβ (Kozlovska et al. 1993, Gene 137:133-137; Ciliens et al. 2000, FEBS Letters 24171:1-4; Vasiljeva et al 1998, FEBS Letters 431:7-11) and MS-2 (WO92/13081; Mastico et al. 1993, Journal of General Virology 74:541-548; Heal et al. 2000, Vaccine 18:251-258) have been produced in bacterial hosts using inducible promoters such as the trp promoter or a trp-T7 fusion (in the case of fr and Qb) or the the promoter using IPTG as inducer substance (in the case of MS-2). The use of inducible promoters is beneficial, to avoid possible toxic effects of the recombinant capsid protein and the metabolic burden of protein expression which both might reduce the growth of the bacterial expression host and, ultimately, the yield of expressed protein.
However, the expression systems used so far for the expression of capsid proteins of bacteriophages have been applied in small scale fermentations, i.e. in laboratory scale and small batch cultures with volumes of typically clearly below 1 liter. An scale up of these systems comprising volumes of 50 liter and more is expected to diminish in a great extent the respective capsid protein yield due to increased promoter leakage and/or lowered plasmid retention.
A further problem associated with commercially desired high-level expression and rapid accumulation of recombinant capsid proteins of bacteriophages is the formation of incorrectly folded protein species and the formation of so called inclusion bodies, i.e. protein aggregates, which are insoluble and which may hamper further downstream processes. Thus, for bacteriophage MS-2 coat protein the formation of protein aggregates and of protein species which lost their ability to self-assemble to VLPs have been reported when the protein was expressed under the control of the strong T7 promoter after IPTG induction using the pET expression system (Peabody & Al-Bitar 2001, Nucleic Acid Research 29(22):e113).
High expression rates of the recombinant capsid protein may therefore have a negative impact on the yield of correctly assembled VLPs. The production of VLP-based vaccines in a commercial scale requires, therefore, the establishment of an efficient, and in particular scalable fermentation process for the expression of recombinant capsid protein of bacteriophages leading to a product of constant quality and purity having the capability of self-assembling into VLPs, whereby the formation of insoluble fractions of the capsid protein is minimised or avoided.
Therefore, it is an object of the present invention to provide a process for expression of a recombinant capsid protein of a bacteriophage which avoids or minimizes the disadvantage or disadvantages of the prior art processes, and in particular, which is scalable to a commercial scale and still leading to a product of constant quality and purity and the capability of self-assemblance to VLPs, and wherein the formation of insoluble fraction of the capsid protein is minimised or avoided.